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A W Boyd

Publications and source records attributed to A W Boyd.

At least 109 records · Page 6Linked to original sources
PubMed1984

Leukemic cell differentiation in vivo and in vitro: arrest of proliferation parallels the differentiation induced by the antileukemic drug Harringtonine.

A variety of chemical agents have been shown to induce differentiation in in vitro cultured neoplastic cell lines. We noted that blast cells in the peripheral blood of acute nonlymphoid leukemia patients treated with the drug Harringtonine appeared to undergo morphological changes that suggested differentiation. In view of the relatively minimal myelotoxicity of Harringtonine, we hypothesized that harringtonine was acting by differentiation-induction, which concomitantly arrested cell division. We tested this hypothesis using two different experimental approaches. First, the promyelocytic leukemic cell line, HL-60, was cultured with Harringtonine and shown to differentiate into a cell, which, by functional cell surface marker and morphological criteria, closely resembled normal monocytes. Furthermore, these changes were accompanied, and indeed slightly preceded by, loss of proliferative capacity. Second, to prove that the leukemic blasts were the cells undergoing the changes observed in vivo, freshly isolated leukemia cell populations were cultured with Harringtonine, and morphological changes paralleling those seen in the patients were observed. Thus, the antileukemic effect of Harringtonine appeared to be due to diversion of the proliferating blast cells into a differentiation pathway, which, as in normal myeloid cells, resulted in the arrest of proliferation.

Acute Disease↗
PubMed1983

Cell cycle kinetic data can be simulated by a simple chemical kinetic model.

This paper develops a model of the cell cycle from the assumption that the rate of progression from one mitosis to the next is limited by the time taken to complete a defined sequence of rate-limiting chemical reactions. The model is shown to be related to earlier models which represent limiting forms of this more rigorous description. Earlier models were classified as stochastic or deterministic, but in this case, while the overall structure is deterministic, the individual steps are stochastically variable. Thus, this description demonstrates that both views were partially correct but were incomplete in detail. By applying the model to several experimental cell cycle curves, sets of coefficients corresponding to reaction rate constants could be obtained. A striking feature of these results is that only a small number (1-3) of slow steps dominated the rate of cell division. A discussion of some recent experimental results suggests possible candidates for the few slow rate-limiting steps, and outlines a rigorous method of testing the model.

Cell Cycle↗
PubMed1983

Analysis of true anti-hapten cytotoxic clones in limit dilution microcultures after correction for "anti-self" activity: precursor frequencies, Ly-2 and Thy-1 phenotype, specificity, and statistical methods.

By the use of split culture techniques we have been able to demonstrate conclusively that the "anti-self" activity and the spontaneous anti-hapten activity within an apparent anti-hapten cytotoxic T cell response is a clonal phenomenon and is not caused by cross-reactivity between anti-self and true anti-hapten clones. With the knowledge of this clonality we have been able formally to prove that the true hapten-generated, hapten-specific response can be obtained by subtracting the response generated by "self" but directed against modified targets from the response generated by modified self and directed against modified targets. Unbiased statistical estimators, which do not make assumptions about the origin (0 responder cells, 100% negative cultures), have been developed to plot accurately limit dilution data (maximal likelihood estimator and minimal chi-square estimator), and the analysis demonstrates that all the components of an apparent anti-hapten response obey zero order (single hit) kinetics. By specifically identifying true anti-hapten and true anti-self clones, we have been able to study the phenotype of their precursors as well as the effectors themselves at the clonal level. Precursors of true anti-hapten and anti-self clones are Thy-1+, Ly-2+. However, anti-hapten and anti-self effector cells show marked clonal variation with respect to Ly-2, as some clones are almost completely insensitive to anti-Ly-2 and complement whereas others show minimal to complete sensitivity. All anti-hapten clones are completely sensitive to anti-Thy-1 and complement, whereas about one-third of anti-self clones show only partial sensitivity, with the most lytic clones showing the most sensitivity. Hapten-specificity and anti-self-specificity have been examined clonally. Ten percent of anti-TNP clones recognize NIP, 10% of anti-NIP clones recognize TNP, and 20% of anti-self clones recognize an allo-target. These figures are in accordance with the overall specificity of effectors generated in bulk culture in the presence of CAS (concanavalin A-stimulated spleen cell conditioned medium). However, hapten specificity and H-2 restriction of bulk generated effectors are improved if CAS is omitted from cultures.

Animals↗
PubMed1982

The effects of a monoclonal anti-mu chain antiserum on the in vitro growth of normal B cells.

Membrane immunoglobulin (Ig) on B cells plays a role in specifically focusing antigen onto the cell surface. An unresolved question is whether the Ig receptor itself has the capacity to deliver a signal to the B cell, and if so what is the mechanism of that signal. To explore these questions we have developed a monoclonal rat anti-mouse mu chain antibody (E4). In this study it is shown that protein A-Sepharose-purified monoclonal anti-mu chain antibody inhibits proliferation of normal B cells in vitro even at the single cell level. This effect was independent of Fc receptor interactions. Furthermore, E4 was shown to inhibit lipopolysaccharide-induced mitogenesis. These results provide evidence that interaction of antibody with only a single determinant on the Ig receptor can deliver a negative signal to the B cell.

Animals↗
PubMed1982

Clonal anergy: the universally anergic B lymphocyte.

The clonal anergy theory of induction of immunological tolerance states that differentiating B lymphocytes that encounter multivalent antigen at the pre-B to B cell transition stage can receive and store a negative signal, which renders them anergic to later triggering stimuli. The theory was tested by using an anti-mu chain monoclonal antibody, E4, as a model tolerogen. The fluorescence-activated cell sorter was used to select B cell-free cell populations from adult murine bone marrow or newborn spleen, and later, to analyze B cell neogenesis in vitro. The presence of E4 at greater than or equal to 1 microgram/ml was required to impede the development of normal numbers of B cells with full receptor status. The subsequent capacity of these B cells to respond in vitro to mitogens was assessed in a filter-cell free microculture system that allows single B cells to proliferate and differentiate. Concentrations of E4 far below those required to affect B cell neogenesis had profound inhibitory effects on the subsequent functional capacity of the B cells. In fact, 10(-3) micrograms/ml of E4 markedly impaired both proliferation and antibody formation, and 10(-1) micrograms/ml, which had no effect on Ig receptor development, abrogated functional capacity. Thus B cells formed in the presence of E4 at 10(-1) micrograms/ml, though possessing the receptor status typical of B cells, were functionally entirely anergic. Exposure to E4 appeared to accelerate the spontaneous death rate of newly formed B cells in vitro. Whether the anergic cell would also have a shortened life span in vivo is not known.

Animals↗
PubMed1982

Two monoclonal anti-human T lymphocyte antibodies have similar biologic effects and recognize the same cell surface antigen.

Two independently produced monoclonal antibodies, UCHT1, an IgG1 antibody, and OKT3, an IgG2 antibody, which specifically identify all human peripheral T lymphocytes, were shown to possess similar functional properties. Both were highly mitogenic to peripheral blood T lymphocytes, and UCHT1 blocked specific T lymphocyte killing of autologous B lymphoblasts when present during either the induction or the cytotoxic phase of the reaction; this, too, is a property reported for OKT3 in an analogous situation. Both antibodies also inhibited the 6-day proliferative response to soluble antigens and mixed lymphocyte culture, although this may be attributable to their strong mitogenicity earlier in culture. These properties were not found with other monoclonal antibodies directed against human T lymphocytes, and suggested that UCHT1 and OKT3 recognize important functional structure(s) on the T cell surface. The identity of the antigen recognized by these two antibodies was demonstrated by 2-directional co-capping, by cold antibody inhibition of binding of radiolabeled antibody, and by immunoprecipitation and two-dimensional gel analysis of labeled T cell surface proteins. Both UCHT1 and OKT3 bound with similar affinities (Ka approximately 2 X 10(9) M-1) to the same number of binding sites (2.8 X 10(4) sites/activated T cell), and both precipitated the same glycoprotein complex with a m.w. of approximately 19,000 in the presence of reducing agent.

Antibodies, Monoclonal↗
PubMed1981

The regulation of growth and differentiation of a murine B cell lymphoma. I. Lipopolysaccharide-induced differentiation.

The B cell lymphoma WEHI 231 has been previously characterized as resembling a resting B cell, bearing large amounts of IgM on the cell surface, but not secreting immunoglobulin. When WEHI 231 cells were cultured together with low concentrations of lipopolysaccharide (LPS) (0.01 to 3 micrograms/ml), increased levels of immunoglobulin were detected in culture supernatants. Analysis by immunoprecipitation and gel electrophoresis demonstrated that this was secreted (19S) IgM. It was noted that small amounts of secretory IgM were produced even in control cultures not deliberately exposed to LPS. Analysis of the cell lysates showed that LPS treatment resulted in a reduction of synthesis of the surface form of IgM. In contrast, the cytoplasmic pool of IgM precursors was considerably expanded by LPS treatment, reflecting the overall increase in synthesis of IgM in LPS-treated cells. These changes in IgM metabolism appear to parallel closely those occurring in normal B cells after mitogen or antigen challenge. This cloned tumor line promises to be a valuable system in which to investigate some of the molecular events in B cell differentiation.

Animals↗
PubMed1981

The regulation of growth and differentiation of a murine B cell lymphoma. II. The inhibition of WEHI 231 by anti-immunoglobulin antibodies.

WEHI 231 is a murine B cell lymphoma, which from our previous studies is highly susceptible to a lipopolysaccharide (LPS) induced differentiation signal. In this paper we show that anti-immunoglobulin (Ig) antibody at low concentrations (0.1 micrograms/ml) inhibits cell proliferation and results in cell death. Heterologous sera specific for mu- and kappa-chains, and a monoclonal antibody (E4) developed in our laboratory and specific for mu-chain, profoundly suppressed the growth of WEHI 231 in both soft agar culture and liquid culture. The F(ab')2 fragments were as potent as intact Ig, indicating that neither Fc receptor binding nor complement activation were required for this inhibition of growth. Neither heterologous antibodies that bound to WEHI 231 but not to the Ig receptor, nor a monoclonal antibody that bound to non-Ig cell surface structures (a brain-associated antigen) inhibited the growth of WEHI 231. LPS did not prevent inhibition of growth by anti-Ig antibody, and even when addition of LPS preceded the addition of anti-Ig, profound inhibition occurred. WEHI 231 thus promises to be a convenient tool for investigating the mechanism of signal transmission by the Ig receptor.

Animals↗
PubMed1980

Mechanism of effector-cell blockade. I. Antigen-induced suppression of Ig synthesis in a hybridoma cell line, and correlation with cell-associated antigen.

A mouse hybridoma cell line, FluIgM-1, which secretes IgM specific for the hapten fluorescein (FLU) was developed to allow detailed analysis of the effector-cell blockade (ECB) phenomenon, in which contact of antibody-forming cells (AFC) with specific antigen results in marked reduction of antibody secretion. Treatment of hybridoma cells with highly substituted FLU conjugates (e.g., Flu20gelatin) resulted in inhibition of plaque formation. The data indicated close parallels with the ECB of normal spleen AFC, both in speed of onset and the dose of antigen required. The inhibition of antibody secretion was confirmed with a biosynthetic-labeling procedure which demonstrated that this was a result of reduced Ig synthesis. The inhibitory effect appeared to be confined to antibody synthesis, in the total protein synthesis, DNA synthesis, and cell-doubling times were unaffected. The association of FLU conjugates with the cells during and following ECB was studied directly using fluorescence microscopy and the fluorescence-activated cell sorter. These experiments showed that FLU conjugates capable of causing blockade aggregated on the cell surface, that the clearance of cell-associated antigen correlated with recovery from ECB, and that at all times when cell associated antigen was detectable, a portion remained bound to the cell surface and was susceptible to enzymatic removal. The latter observations supported previous findings suggesting that ECB was mediated by extracellular antigen. The direct observation of aggregates of antigen on the surface of blockaded cells is consistent with a mechanism involving cross-linking of Ig receptors. Finally, Fc receptors were not present on hybridoma cells, excluding their involvement in induction of ECB.

Animals↗
PubMed1979

A case of paroxysmal nocturnal haemoglobinuria terminating in a myeloproliferative syndrome.

This report discusses the case of a 60-year-old man who presented in 1969 with thrombocytopenia and mild marrow hypoplasia. A diagnosis of paroxysmal nocturnal haemoglobinuria (PNH), was established. The subsequent course included episodes of overt intravascular haemolysis. Thrombocytopenia reverted on several occasions during Oxymetholone therapy. The terminal phase of the illness was marked by the development of a leukocytosis and densely hypercellular bone marrow with splenomegaly. The features were those of a myeloproliferative disorder, although frank leukaemia did not develop.

Alkaline Phosphatase↗
PubMedDate not supplied

Characterization of two novel pre-B-cell lines (LK63 and LiLa-1): potential models of pre-B-cell differentiation.

In this report we describe two newly isolated pre-B acute lymphoblastic leukaemia cell lines. Both cell lines lack EBV as detected by the EBNA-1 gene probed Southern-blots. Neither cell line expressed the B-cell-specific CD20 antigen on the cell membrane. However surface expression of CD20 was induced by phorbol ester (TPA) on both LiLa-1 and LK63 cell lines. Other pre-B and B-cell lines, such as Reh, Nalm-1, and BALL-1 did not exhibit these changes in phenotype. Previous immunoprecipitation studies have noted that a broad 50-55 kD band co-precipitates with the characteristic 33-37 kD CD20 protein. We demonstrate that, while the 33-37 kD CD20 species was undetectable on resting LiLa-1 and LK63 cells, in each case a 50-55 kD protein was immunoprecipitated by the CD20 antibody. However, the failure to detect any cell surface CD20-associated antigen on the control cells by immunophenotyping indicated that the CD20 epitope of the 50-55 kD molecule was not expressed on the cell surface. Following exposure to TPA the 50-55 kD species was reduced over 48-72 h while the level of the p33-37 CD20 protein was increased. Northern-blot analysis showed that the 50-55 kD protein was not a cryptic form of CD20 as the uninduced cells contained no detectable CD20 mRNA. The decrease of the 50-55 kD protein and the acquisition of the mature CD20 molecule were paralleled by a decline in proliferative activity in both cell lines. As expression of CD20 by normal pre-B cells also coincides with the cessation of cell division and maturation towards a mature B-cell phenotype, these cell lines appear to represent models for a discrete stage of B-cell differentiation which may be valuable in defining the signals regulating pre-B-cell proliferation.

Antigens, CD↗